The role of ascorbic acid (Asc) in the biosynthesis of both catecholamine neurotransmitters and neuropeptide hormones and thus, in overall neuroendocrine functions has been well recognized. Identification of a complex and intricate ascorbate regenerating system (ARS) in neuroendocrine granules further elaborate the central role of Asc in neuroendocrine functions. Although, the recent efforts have been directed towards the understanding of the functions of Asc/ARS in relation to catecholamine neurotransmitter and neuropeptide hormone biosynthesis, progress of these efforts has been hampered by the complexity and the diversity of the effects of intra- and extra-cellular factors. The work proposed herein is focused on the understanding of the role of Asc/ARS in catecholamine neurotransmitter biogenesis and regulation by systematic investigation of the two major Asc-dependent systems, dopamine beta-monooxygenase (DbM) and cytochrome b561, at the molecular level. In the initial phase of our program, we will develop a sensitive, continuous spectrophotometric assay for DbM and extend it to measure the electron flux into neuroendocrine granules via cyt b561 using adrenal chromaffin granule ghosts as a model system. In the second phase, we will develop various Asc-based chemical probes, i.e. alternate electron donors, competitive and suicide inhibitors, multi-substrates/inhibitors, active site directed inhibitors, and photoaffinity labels to examine the Asc site of DbM in detail. The knowledge gained by these studies will be used and extended to examine the chemistry and the specificity of cyt b561 using purified reconstituted systems and then extend it to more complex cyt b561-mediated ARS in chromaffin granule ghosts. In the third phase, we will use the above assay as well as the Asc-based chemical probes to examine the effect of extra- and intra-cellular factors on norepinephrine biosynthesis in granule ghosts and intact chromaffin granules, in order to understand the molecular mechanisms and interrelations between the catecholamine transporter, membrane ATPase, both soluble and membrane bound DbM and the electron transport protein, cyt b561. The results of these studies will yield information which will be valuable in understanding the crucial role of Asc/ARS, in the dynamics of adrenergic neurosecretory vesicles. In addition, these findings could probably be extended into the peptidergic neurosecretory vesicles, since the peptide processing terminal enzyme, peptidyl amidating monooxygenase (PAM), is also Asc-dependent.